The interaction of eukaryotic DNA dependent RNA polymerase II (wheat germ) with Adenovirus 2 DNA was studied. Polymerase II can bind to virus DNA and form two types of stable binary complexes: (a) I complex from which initiation cannot occur and (b) RS complex from which initiation of RNA synthesis can occur when nucleoside triphosphates are present in reaction. The formation of all RS complexes is preceded by the formation of I complexes. The formation of RS complexes exibit a characteristic sigmoidal dependence on temperature. The transition from I to RS complex results in the opening of 4 to 5 base pairs in DNA. The association constant of formation of binary complexes was determined to be on the order of 1 x 10 to the 8th power per second per male and dissociation constant was 1 x 10 to the minus 4th power per second. The equilibrium constant (Keq) is equal to 1 x 10 to the 12th power M -1, and is similar to the value obtained for E. coli DNA dependent RNA polymerase. Stable complexes are formed at 6 to 7 sites on the adenovirus 2 DNA molecule. These sites have been mapped using "proteinless" EM procedure spreading of DNA molecules (Vollenweider et al. 1976). The position of complexes on DNA molecule are: 5.1 1.2, 13.0 1.4, 32.0 1.5, 45.4 2.4, 70.6 2.2, 88.5 2.2 and 96.4 0.5. In a standard enzyme preparation only 10 to 20% of the molecules of enzyme are able to form stable binary complexes with double-stranded adenovirus DNA. The remaining enzyme can bind and initiate RNA synthesis only on single-stranded DNA.